Department of Agriculture and Food Brain removal techniques The conventional method for brain removal involves sawing through the bone of the dorsal cranium and removing a cap of bone to expose the brain. Two alternate methods are shown below that, with practice, can be less tiring and faster to perform. The brains removed by these techniques provide a sample suitable for routine diagnostic and transmissible spongiform encephalopathy (TSE) exclusion testing. Method 1: Longitudinal craniotomy Method 2: Transverse craniotomy When using the transverse craniotomy technique in cattle, it is advisable to leave the head attached and immobilise for sawing by tensioning nose grips to a vehicle or other solid object. Alternatively, flex the neck and tie the nose grips to the hock. In small ruminants, removing the Step 3: Turn the head over and cut through the head is recommended. soft and hard palate and ventral cranium. Step 3: Begin with the rostral half of the skull. Using curved scissors, cut the ventral nerve roots and the olfactory bulbs. Step 1: Remove the head at the atlanto-occipital Remove the rostral brain. joint. Remove the tongue and pharynx to expose the hard and soft palate. Split the mandibular symphysis and place the head on a non-slip surface such as a rubber mat or onto the ground, using the separated mandible to improve stability. Step 6: Using scissors, cut the cranial nerves on the ventral and lateral surfaces inside the cranium Step 1: Stabilise the head for and then around the cut spinal sawing by placing it on a non-slip cord at the foramen magnum. surface or mat. The head does not Using a finger or plunger from a Step 4: Stand the head upright on the foramen need to be skinned, but a knife cut syringe, gently push the hindbrain magnum and use a knife to sever any remaining through the skin at the intended and cerebellum rostrally out of the attachments. Lever the nasal bones apart, using line of sawing is recommended. cranium. the hatchet to increase leverage and split the skull. Step 4: Shell out the occipital If nasal bones feel as if they will snap, check all lobes in the caudal half of the skull the bone is cut, especially between the occipital to expose the opaque tentorium condyles. cerebelli. Step 2: Using a hatchet and a mallet, make a dorsal midline cut through the bone from the nose to the foramen magnum. Place hatchet blade at cutting site and use mallet to split the bone. Do not swing the hatchet. The head does not need to be skinned, Step 7: Fix the three brain but making an incision through the skin along the Step 2: Begin sawing vertically segments whole in 10% buffered Use scissors to cut the remaining meninges proposed line of cutting helps to stop the hatchet Step 5: in a line 1cm rostral to external formalin. Note: for TSE exclusion and cranial nerve attachments to allow removal of the blade from deviating. ear canal and extend the cut testing, remove the dorsal entire brain. Note: for TSE exclusion testing, remove through the bone until the cranium cerebellum (sheep only) and 2-3cm the dorsal cerebellum (sheep only) and 2-3cm spinal hinges apart. Remove the head Step 5: Cut the tentorium to spinal cord and submit these cord and submit these sections fresh. Fix remainder at the atlanto-occipital joint if still expose as much of the cerebellum sections fresh. Fix remainder of of brain in 10% buffered formalin. attached. as possible. brain in 10% buffered formalin. Important Disclaimer: The Chief Executive Officer of the Department of Agriculture and Food and the State of Western Australia accept no liability whatsoever by reason of negligence or otherwise arising from the use or release of this information or any part of it. Copyright © Western Australian Agriculture Authority, 2015 Department of Agriculture and Food Brain sampling tips for TSE exclusion testing Brain samples required for Brain sampling do’s: Brain swabbing methods that 3 Take care not to damage key TSE brain sites when removing the National TSE Surveillance the brain and taking the fresh samples (see Figure 2). keep the brain intact 3 Use enough 10% buffered formalin and a sufficiently large Program include: histology pot so the brain does not fix in a distorted position: Method 1: For most meningitis cases it is suitable to swab • sheep brain – use a 1L pot filled to the top with formalin the base of the brain. Sheep: • cattle brain – use a 2L pot filled to the top with formalin. 3 Allow the brain to fix in the formalin pot at room temperature. 1. fresh dorsal cerebellum 3 Check the case meets the TSE eligibility criteria (listed on the 2. fresh spinal cord, 2–3cm in length TSE lab submission form). Cows nine years of age or older 3. fix the rest of the brain and brainstem whole. are ineligible. Cattle: Brain sampling don’ts: 1. fresh spinal cord, 2–3cm in length 7 Don’t split the brain in half lengthways (longitudinally) as this 2. fix the rest of the brain and brainstem whole. damages TSE sites. 7 Don’t submit a half fixed/ half fresh brain. To culture, use a swabbing technique that will keep the brain intact (see below). 7 Don’t remove the fresh spinal cord sample from too close to the cerebellum – imagine a perpendicular line behind the 1. fresh dorsal cerebellum (no 2. fresh spinal cord, more than dorsal one-third of 2–3cm in length – cerebellum and avoid sampling on the cranial side of the line. cerebellum) – sheep only cattle and sheep 7 Don’t remove more than one third of the dorsal cerebellum when removing the fresh cerebellum sample in sheep. 7 Don’t put pots containing tissue and formalin in the fridge or freezer. Method 2: Listeria can be recovered by stabbing a swab through the dorsal cerebellum into the brainstem. The swab must be angled in a cranial direction so it does not damage the obex. Take care not to damage the obex Figure 2 Key brain sites for TSE exclusion Figure 1 Collection of fresh brain TSE samples Important Disclaimer: The Chief Executive Officer of the Department of Agriculture and Food and the State of Western Australia accept no liability whatsoever by reason of negligence or otherwise arising from the use or release of this information or any part of it. Copyright © Western Australian Agriculture Authority, 2015.